利用体外基因敲除技术检测小鼠胚胎成纤维细胞（mouse embryonic fibroblasts，MEFs）周期和迁移能力的变化，探究蛋白磷酸酶2A 的 Cα亚基在 MEFs 细胞迁移过程中的作用。用 Cαfl／fl的纯合子雄鼠与雌鼠1∶2配对，取 E12．5 d 的胚胎制作小鼠胚胎成纤维细胞。利用表达 Cre 重组酶（Ad-Cre-EGFP）以及GFP 荧光蛋白（Ad-EGFP）的腺病毒载体，对 P3代 MEFs 进行感染，分别用 PCR，RT-PCR 和 Western blot方法进行基因型鉴定。同时利用流式分选技术检测感染后 MEFs 的细胞周期的变化，利用细胞划痕试验检测了其细胞迁移能力的变化。结果显示，胰酶消化的方法成功获得了小鼠胚胎成纤维细胞，DNA，RNA 和蛋白水平的鉴定获得了3对3的野生型和基因敲除 MEFs。流式分选发现 Ad-Cre 处理的 MEFs 与 Ad-EG-FP 处理的 MEFs 相比，处于 G2期的细胞比例为30．8％±2．57％，高于 Ad-EGFP MEFs 的23．9％±2．46％，并且细胞碎片的比例也远高于后者。划痕试验表明，Cα亚基缺失后细胞迁移能力下降，12 h 就显著低于对照组。结果表明，腺病毒携带的 Cre 重组酶能够在体外有效地敲除掉 MEFs 中的蛋白磷酸酶2A 的Cα亚基，PP2A Cα亚基的缺失会导致 MEFs 细胞周期倾向于阻滞在 G2期，并会降低细胞迁移的能力。%To investigate the effects of catalytic subunit α of protein phosphatase 2A (PP2A)on cell cycle and cell migration in mouse embryo fibroblasts(MEFs),the mouse embryos were got at E12.5 by mating the heterozygotes of Cαsubunit conditional knockout male and female homozygous mice at the ratio of 1∶2.MEFs were prepared by trypsin digestion and genotyped by PCR,RT-PCR and Western blot to identify the genetic basis of each embryo.Adenovirus associated Cre recombinase and GFP immunofluorescence protein were constructed and delivered to the P3 MEFs to knockout the Cαgene,GFP was used as control. After confirming the knockout efficiency by PCR,RT-PCR and Western blot,FACs was carried out to de-termine the cell cycle.Cell scratch test was carried in MEFs treated with the two kinds of viruses to inves-tigate the effects on migration.Adenovirus associated Cre recombinase could knockout the gene efficiently in vitro .The knockout MEFs at G2 stage was 30.8% ± 2.57%,while the control MEFs was 23.9% ± 2.46%,and had more cell debris.Loss of Cαdecreased the cell migration ability significantly from 12h after the scratch.Cre recombinase delivered by adenovirus could efficiently pop out double floxed gene in vitro . Disruption of Cαsubunit slightly arrested the MEFs at G2 stage,and decreased the migration ability.
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